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Abstract

Coherent control can be used to selectively enhance or cancel concurrent multiphoton processes, and has been suggested as a means to achieve nonlinear microscopy of multiple signals. Here we report multiplexed two-photon imaging in vivo with fast pixel rates and micrometer resolution. We control broadband laser pulses with a shaping scheme combining diffraction on an optically-addressed spatial light modulator and a scanning mirror allowing to switch between programmable shapes at kiloHertz rates. Using coherent control of the two-photon excited fluorescence, it was possible to perform selective microscopy of GFP and endogenous fluorescence in developing Drosophila embryos. This study establishes that broadband pulse shaping is a viable means for achieving multiplexed nonlinear imaging of biological tissues.

Figures (6)

Top (a) and side (b) views of the pulse shaper. A spatial light modulator (SLM) is placed in the Fourier plane of a grating (GR)-cylindrical mirror (CM) combination in a folded 4-f configuration. The 2D phase mask is used in the x-dimension as a phase shaper and in the y-dimension as a ruled grating used in Littrow configuration. A galvanometer mounted mirror (G) switches the light beam between the two areas of the SLM with two different phase shapes.

2D masks as viewed in the beam propagation direction. In panel (a), a mask with uniform grating length is shown and the resulting angular chirp is shown in (c). The angular chirp was corrected by using a grating length directly proportional to the wavelength as shown in (b). Panel (d) schematically illustrates that the angular chirp is removed due to the scaled grating.

(a) Trace obtained from a chirp scan measurement for a 0.8NA 40× water immersion objective in the case of a residual third-order phase. (b) Second order interferometric autocorrelation trace (line) and the intensity autocorrelation trace (circles) corresponding to a near transform limited pulse (~14 fs) at the focus of the microscope objective. (c) Laser spectrum (L.S.) at the focus of the microscope objective and Spectral phases (R.S.-red shifted and B.S-blue shifted) for the two different pulse shapes used in the imaging experiments. The “red shifted” spectral phase is anti-symmetric with respect to frequency corresponding to λ=840 nm and the “blue shifted” spectral phase is anti-symmetric with respect to frequency corresponding to λ=780 nm. (d) Two-photon spectra measured at the focus of the microscope objective for a near transform limited pulse (TL), “blue shifted” spectral phase and “red shifted” spectral phase in (c).

(a and b) Images of 100 nm beads located (a) at the center and (b) at the edge of the field of view respectively. The scale bars correspond to 1 µm. Each image is obtained by merging the two images acquired by the red-shifted (shown in green) and the blue-shifted (shown in blue) pulses. (c), (d): radial intensity point spread functions measured from the bead images shown in (a) and (b). FWHM is 0.49 µm. (e), (f): axial intensity point spread functions measured from the same beads. FWHM is 2 µm.

Multiplexed in vivo imaging of an eGFP-expressing Drosophila embryo using (a) blue-shifted pulse shaping for preferential excitation of the endogenous fluorescence at 780 nm and (b) red-shifted pulse shaping for preferential eGFP excitation at 840 nm. Dorsal side is up. (c), (d): linear combinations of images (a) and (b) for separating the two fluorescent components (spectral unmixing). (e), (f): images obtained by combining (c) and (d) at two different stages of embryo development. Tissue extension is visible at the embryo posterior pole during germ band extension (white arrow). (g), (h): Kymographs (space-time projections) along the dotted red lines indicated in (c) and (d). These projections reveal the correlated motion of cells (h) and underlying yolk structures (g) during extension movements. (i) and (Media 1) 3D+t movie of the posterior region of another embryo imaged from the dorsal side, during germ band extension. One 3D image was recorded every minute. Scale bars: 50 µm (X) and 5 min (time).